HK1118703B - Production of glucosamine from plant species - Google Patents

Description

Production of glucosamine from plant species

Technical Field

The present invention relates to a method for producing a plant material containing glucosamine at a level equal to or higher than 0.5% (wt) dry matter.

Background

Use of glucosamine

The use of pure glucosamine in joint diseases is widely described in the patent and scientific literature, usually in combination with other compounds and extracts of various natural sources. Pure glucosamine is added as glucosamine hydrochloride or glucosamine sulfate and results from shellfish hydrolysis. For example, WO2000/0074696 describes "a herbal composition for treating inflammation or degeneration of joint tissues such as arthritis, comprising glucosamine and tripterygium wilfordii, Ligustrum lucidum and/or erysipelas, wherein pure glucosamine is mixed with a plant product. Other patents relate to compositions of plant carbohydrates as dietary supplements (EP 1172041 or EP 923382), in which the glucosamine is derived from chitin, that is to say, in turn, from shellfish hydrolysis.

The use of glucosamine as an anti-osteoarthritis agent has been intensively developed during the last decade. Glucosamine is presumed to be the only active compound in joint diseases such as osteoarthritis (until now, only symptomatic treatments such as non-steroidal anti-inflammatory drugs have been sought effective).

Glucosamine has also been shown to prevent cartilage degradation by inhibiting the production of MMPs (matrix metalloproteinases) such as MMP1, MMP3 and MMP 13. Significantly, glucosamine is also associated with the aging process of the skin, which is characterized mainly by a continuous loss of elasticity and by a loss of water. Skin aging is reflected by major structural and compositional changes. Most notably, aged skin has less collagen and glycosaminoglycans (glycosaminoglycans) compared to young skin. Glycosaminoglycan molecules produced by the skin include hyaluronic acid (poly (d-glucuronic acid-n-acetyl-d-glucosamine)), chondroitin sulfate, and dermatan sulfate. Skin cells produce higher amounts of hyaluronic acid in response to exfoliation. Hyaluronic acid has a great hydration capacity.

Inhibition of MMP-1 is associated with inhibition of degradation of the polysaccharide/collagen and therefore also with skin ageing: MMP-1 can be induced by UV light and is considered to be a marker for skin aging. The invention of US 2002/119107 is based on the selective inhibition of MMP-1, and claims topical compositions for protecting human skin from collagen degradation. US 2004/037901 claims therapies for inhibiting adverse signs of the effects of skin aging comprising rosemary plant extracts which inhibit the expression of metalloproteases.

Glucosamine has been shown to significantly improve skin dryness and exfoliation. Glucosamine increases the moisture content of the skin and improves the smoothness of the skin. These findings suggest that long-term intake of glucosamine is effective for improving moisture content and smoothness of skin.

Oral supplement containing glucosamine has been shown to cause a reduction in the number of visible wrinkles (34%) and a reduction in the number of fine lines (34%) in a group of women taking the supplement. The use of oral supplements containing glucosamine, minerals and various antioxidant compounds can potentially improve the appearance of visible wrinkles and fine lines.

US 6413525 describes a method of substantially exfoliating skin. In particular, the invention relates to topical compositions containing an amino sugar in the form of N-acetylglucosamine: when young skin cells are exposed after shedding, they produce a greater amount of hyaluronic acid, which is a glycosaminoglycan composed of alternating repeating chains of dextrorotatory glucuronic acid and N-acetyl-dextrorotatory glucosamine. N-acetyl-D-glucosamine is known to be the rate-limiting factor in the production of hyaluronic acid by living cells. Topical application of glucosamine helps in the sustained production of hyaluronic acid.

Other topical compositions containing N-acetyl-d-glucosamine are also disclosed, for example, in JP 59013708 (softening and moisturizing skin) or in us patent 5866142 (compositions for exfoliating skin).

Sources of glucosamine

Glucosamine, 2-amino-2-deoxy-D-glucose, is a naturally occurring fructose derivative and is a major component of glycoproteins and proteoglycans, an important component of many eukaryotic proteins. It is the main component of mucopolysaccharide (mucopolysaccharide) and chitin. Glycosaminoglycans (mucopolysaccharides) are large complexes incorporated into connective tissue, skin, tendons, ligaments, and cartilage.

Industrial sources of glucosamine

Industrial glucosamine is a pure compound obtained by acid hydrolysis of chitin from crustaceans (a complex carbohydrate derived from N-acetyl-D-glucosamine). For example, us patent 6486307 describes an improved process for hydrolysis of chitin acids: a process for producing glucosamine hydrochloride by milling chitin to a very fine size and digesting with concentrated hydrochloric acid.

Glucosamine can also be produced by enzymatic hydrolysis of crustaceans. For example, U.S. Pat. No. 5998173 describes a novel process for the direct production of N-acetyl-D-glucosamine from chitin by hydrolyzing chitin from crustaceans using a complete set of enzymes of the chitinase family.

Patents have also been filed which protect microbial fermentation processes in which cultured microorganisms biosynthesize glucosamine. For example, US 6372457 describes methods and materials for producing glucosamine by fermentation using genetically modified microorganisms.

All of these methods involve competing with crustacean extracts to produce pure extracted glucosamine.

GB 649791 relates to improvements in dried chicory (chicory). The method of this patent comprises the steps of:

-cutting the roots of chicory,

-fermenting chicory roots under aerobic conditions at a temperature substantially not exceeding 70 ℃ (optimal temperature is 50 to 55 ℃) for 7 to 8 hours, and

quick drying of chicory at 150 ℃ for about 30 minutes.

These conditions are not capable of producing glucosamine.

Carvalho et al in a publication entitled "inulin production by y Vernonia herbacea inhibition and time of prevention" (Revista brasilerad botanica, 1998) show that the addition of fertilizer during the cultivation of plants containing inulin does not increase the inulin content.

In WO2005/053710 it was found that glucosamine can be formed from several plant materials according to a special drying process, whereby glucosamine contents of 150 to 1000mg per kg dry weight are obtained.

Summary of The Invention

In a first aspect of the invention, a novel process for obtaining glucosamine from plants is described, using a nitrogen-based fertilizer (nitrogen-based fertiliser) prior to harvesting the plant material and subjecting it to a heat treatment. The nitrogen-based fertilizer acts as precursor of glucosamine to obtain a plant material containing glucosamine at a level higher than 0.5% dry weight (5g/kg dry weight). The present invention thus enables glucosamine contents to be obtained in plant materials higher than previously described in the prior art (as reflected in WO 2005/053710). Thus, less plant material or plant extract is required to obtain the active dose of glucosamine described in the literature. Therefore, the method is more useful on an industrial scale.

The above-described fertilizers can be added in the field or in hydroponic culture systems several hours up to several weeks before harvesting the plant material.

Detailed Description

In the present description, the term "heating" (and thus derived "heated") is to be understood as a heat treatment at a temperature in the range of 70-110 ℃ for more than 10 hours, preferably less than 1 week. This heating process may be described as a drying process. The heat treatment may also be present in the form of liquid impregnation carried out under the same temperature and time conditions, instead of the drying treatment.

In the present description, "free glucosamine" is understood to mean non-polymeric glucosamine.

In the present description, "high amount of glucosamine" is understood as meaning an amount of glucosamine higher than traces of glucosamine, higher than the amount in the corresponding fresh (not dried) material, and higher than any content cited in the literature or in the patent. It is understood that glucosamine is present in an amount higher than 0.5% per kg dry matter of the raw material.

In this specification, "plant" and "plant material" are considered synonyms. By "plant", "plant material" or "plant extract" is understood any plant material capable of producing glucosamine according to the heat treatment of the present invention, as well as any type of plant extract capable of producing glucosamine from said plant material by any extraction method known to the skilled person, according to the heat treatment of the present invention. For example, a plant containing an amount of glucosamine can be a dried or rehydrated plant material that has undergone the process of the invention. The plant extract containing an amount of glucosamine may be an aqueous solution extracted from said plant subjected to the treatment of the invention.

Thus, in one aspect, the present invention describes a novel method for obtaining glucosamine from plants.

With regard to the first object of the present invention, plants or plant extracts are treated according to the invention so as to contain high amounts of natural free glucosamine.

In a preferred embodiment, the plant or plant extract is from any part of the plant, such as a leaf, stem, fruit, seed, root, grain or cell culture. After the controlled heat treatment of the plant material, the plant or plant extract may be in the form of a dried, lyophilized extract of leaves, roots and/or fruits depending on the plant source, or a fresh plant, or a glucosamine-rich fraction.

In one embodiment, the cultivation of the plant species is done in the field or in a hydroponic culture system.

In a preferred embodiment, the cultivation of the plant species is done in a hydroponic system.

Selecting a plant or plant extract capable of producing free glucosamine by the method of the invention; in particular, it may be selected from plant species containing sucrose, fructose or inulin, such as Cichorium (Cichorium), Daucus (Daucus), Helianthus (Helianthus), Beta (Beta).

For example, in one embodiment, the plant material or plant extract may be derived from, for example, chicory (Cichorium intybus) root, carrot (Daucus carota) root, Jerusalem artichoke (Jerusalem turcoshoke) stem, beet (Beta vulgaris) root.

In a preferred embodiment, the plant species is chicory, for the production of Belgian endive (Belgian endive) in the field or in hydroponic systems: namely chicory (chicon), witloof (witloof chicory), chicory (witloof), French chicory (French end), white endive (whitement end), parsley (Dutch chicory), chicory (succinory), common chicory (common chicory) or dandelion (Italian dandelion). "witloof chicory" is a common name used by most horticulturists of cultivated plants, whereas "belgian chicory" is more commonly used as the end product sold to consumers in grocery stores. Endive is the secondary growth of the top bud of chicory root. One growth occurs on field plants grown from seeds. Secondary growth does not occur in the field, and usually occurs in the dark in the factory. Each root with one main bud causes the development of chicory (endive).

In a most preferred embodiment, the culturing of chicory is done in a hydroponic system. Chicory roots are placed in a recirculating medium and nutrients in the nutrient solution, including the fertilizer responsible for the formation of more glucosamine, promote the growth of feeder roots that germinate from the bottom of the chicory root. These feeder roots act as pumps throughout the chicory root vasculature. That is, the skilled artisan will recognize many variations of this example to cover a wide range of treatments and mixtures to rationally adjust the naturally occurring levels of the compounds of the invention for a variety of applications.

In one embodiment, the endive production system herein is used in the presence of a fertilizer acting as glucosamine precursor, wherein the chicory roots are harvested simultaneously with the aerial parts (endives).

In another embodiment, after the commercial production of endives herein, the chicory roots are again placed under hydroponic conditions in the presence of a fertilizer acting as glucosamine precursor.

In one embodiment, fresh plant material treated according to the invention, or plant material treated according to the invention and then dried and subsequently rehydrated, can be used as starting material for processing to obtain plant material with a high glucosamine content according to the invention.

In a preferred embodiment, fresh plant material is used.

According to the present invention, plants were cultivated in various ways using nitrogen-based fertilizers acting as precursors of glucosamine, obtaining a much higher amount of glucosamine than WO2005/053710 (higher than 5g/kg dry matter of chicory roots).

As disclosed in WO2005/053710, the drying process described therein is one way to obtain glucosamine in large quantities in plants: glucosamine levels that can be obtained using the drying process described in WO2005/053710 are: 500mg/kg dry matter of chicory root, 100mg/kg dry matter of carrot root or 50mg/kg dry matter of jerusalem artichoke stem or beetroot.

Fresh, dried or rehydrated plant material previously contacted during cultivation with a nitrogen-based fertilizer is heated by liquid immersion or drying at a temperature below 110 ℃, preferably at a temperature of 70 to 110 ℃, most preferably at a temperature of 70 to 91 ℃ or less for more than 10 hours, preferably less than 1 week, preferably 10 to 120 hours, e.g. 12 to 50 hours, depending on the plant species and plant organs. If the temperature is too low and/or the heating time is too short, glucosamine will not be efficiently produced or will be produced too slowly, rendering the process economically unfeasible. Conversely, if the temperature is too high and/or the heating time is too long, glucosamine is produced but is then gradually degraded.

Therefore, in order to obtain a glucosamine content of at least 5g glucosamine/kg corresponding to the dry matter content of the heat-treated plant material, it is necessary to select the temperature and the time.

Most preferred examples include drying in an oven at a temperature of 85 ℃ for 48 to 72 hours.

The fertilizer according to the invention is a compound capable of forming a sugar-nitrogen compound condensation reaction, which is necessary for the formation of glucosamine. Preferably the fertilizer is in the presence of an ammonium salt. Examples of such ammonium salts are ammonium nitrate or ammonium sulfate and the like. The preferred glucosamine precursor is ammonium sulfate, which shows surprisingly good results in the process according to the invention.

Fertilizers are used for hours to up to weeks before harvesting the plant material. That is, the skilled person will recognise many variations of this example to cover a wide range of fertiliser applications to rationally adjust the naturally occurring levels of the compounds of the invention for a variety of applications.

With regard to the final treatment leading to glucosamine formation, a suitable method for preparing plant material is described in WO 2005/053710: plant material is harvested, cut and dried in an oven or industrial dryer at a temperature below 110 ℃, preferably between 80 and 105 ℃, optimally 91 ℃ or below, for more than 10 hours, preferably less than 1 week, preferably between 10 and 120 hours, e.g. between 12 and 50 hours, depending on the plant species and plant organs. While not wishing to be bound by any theory, it is believed that it is preferred to cut the plant material into pieces or clumps, preferably with a maximum width of 5 mm. The inventors do believe that this is important to the present invention in order to achieve the optimal thermodynamic exchange.

The addition of a fertilizer acting as precursor of glucosamine prior to harvesting of the plant material enables a significant increase of the above reaction, in the absence of precursor, of only a few hundred milligrams of glucosamine per kilogram of dry matter, up to at least 5g glucosamine per kilogram of dry matter of the respective plant material.

The process of the present invention directly produces glucosamine in free form. Without wishing to be bound by any theory, it is believed that the process produces at least half of the glucosamine in free form, and even produces almost all of the glucosamine in free form. Indeed, it is believed that at least 50%, at least 70% and even at least 90% of the glucosamine produced according to the process of the invention is in free form. This is a further advantage of the present invention, in contrast to the known techniques for producing glucosamine, where a hydrolysis step is mandatory, for example, in order to release free glucosamine from complex molecules such as chitin, glycoproteins or proteoglycans.

The plant or plant extract according to the invention can be used for the preparation of a food composition without further treatment or extraction. The composition may be in the form of a nutritionally balanced food or pet food, a dietary supplement, a therapeutic or a pharmaceutical composition.

Examples

The following examples are illustrative of some of the products and methods of making the products within the scope of the present invention. They should not be construed as limiting the invention in any way. Changes and modifications can be made to the invention. That is, the skilled artisan will recognize many variations of these examples to cover a wide range of formulations, ingredients, treatments, and mixtures to rationally adjust the naturally occurring levels of the compounds of the invention for a variety of applications.

Example 1:

fresh chicory roots treated with fertiliser during the hydroponic production cycle of chicory (endive)

The method for producing endive is adopted to culture chicory root under the condition of solution culture at 20 ℃ and 80% relative humidity in dark for 21 days. The nutrient solution contained 0.1M ammonium sulfate. After 21 days, chicory and roots were harvested. The roots were cut into pieces (0.5X 0.5cm) and then dried in an oven at 91 ℃ for 40 hours.

And (3) analysis:

extraction of glucosamine

2g of ground and dried chicory root are extracted with 20ml of water at room temperature for 1 minute. In Schleicher&The solution was filtered on a Schultz (n ° 597) filter or centrifuged. The solution purification step was carried out using a cation exchange column (Oasis cartridge WATERS, MCX type, ref.186000776). With 2% (v/v) MeOH/NH₄The OH elutes the basic compound captured on the matrix. After filtration, aliquots were injected directly onto the LC system (DIONEX). Separation:

analysis was performed by HPAE/PED system using ion exchange PA1 column (4 x 250mm) with DIONEX DX500 apparatus.

The procedure is as follows:

elution (%)

Time (minutes)	H2O	0.1 M NaOH	0.25 NaOH	Note
					0	85	15	0	Balancing
60	85	15	0
					60.1	0	0	100	Washing machine
70	0	0	100

70.1	85	15	0	Balancing
					90	85	15	0

Flow rate: 1 ml/min. Injection volume: 20 μ l. The standard is as follows: glucosamine from Sigma (ref: G4875).

Under these conditions, glucosamine has a retention time of about 11 minutes, which is easy to detect, for further quantification of suitably processed chicory extracts. The concentration quantified by this method in this example was 5000mg/kg dry weight, in contrast to less than 900mg/kg without precursor, whereas less than 10mg/kg without heat treatment or commercially available dried chicory root.

It is therefore possible to carry out the invention on plant materials which produce endives as a valorized by-product. Confirmation of the Presence of glucosamine:

to confirm the presence of glucosamine in chicory plant extracts, three different qualitative techniques were evaluated.

Thin Layer Chromatography (TLC)

Pure glucosamine and plant extracts were analyzed on HPTLC (high Performance thin layer chromatography) silica gel plates (Merck, ref.1.05642) with ethyl acetate/MeOH/water (50/50/10; V/V/V) as eluent. After elution, the silica gel plate was sprayed with a 1% ninhydrin acetic acid solution and heated at 120 ℃ for 10 minutes. The reference and extract appeared as pink/blue spots at the same rate factor (Rf).

Chemical degradation

In the presence of ninhydrin, the glucosamine undergoes oxidative deamination, resulting in the release of arabinose, which can be easily detected by conventional sugar LC analysis. The presence of arabinose in the control and chicory extracts was clearly confirmed.

Derivatization of glucosamine

Such as Zhongming et al: "Determination of nucleotides, gluconamide hydrochloride in raw materials, assay for and plasma using pre-column chromatography with UV HPLC. in J. of pharmaceuticals. and biomed. analysis, 1999(20), 807-814" reverse phase chromatography of pure compounds and plant extracts using a pre-column derived from phenyl isothiocyanate (phenylisothiocyanate) and detection by UV light (. lamda. 254 nm).

Peaks corresponding to derivatized glucosamine were detected in both the pure compound as well as in the chicory extract.

Mass spectrometric analysis

Plant extracts were analyzed by electrospray mass spectrometry in positive ionization mode to confirm the presence of glucosamine. The mass spectrometer was a time-of-flight instrument (LCT from Micromass using a Z-spray interface). Standard glucosamine gives an ion with m/z 180.0887. This ionic fragment is found in the plant extract analyzed.

Example 2:

fresh chicory roots treated with fertiliser after a productive period of hydroponic chicory (endive) culture

After harvesting endives cultured under the conditions herein for normal commercial production, the remaining roots are subjected to the same treatment. The roots were again placed in the same hydroponic conditions as in example 1 and a second cultivation cycle was carried out with a nutrient solution containing 1M ammonium sulfate.

On day 5, roots were harvested and then treated (cut, dried) as described in the examples. As a result, the concentration of glucosamine was 10g/kg dry weight.

This is another example of the possibility of implementing the invention on plant materials which produce valorized by-products of endives.

Claims (13)

1. A method for producing glucosamine from plants, wherein fresh plant material or rehydrated dried plant material or plant extract is heated at a temperature of 70 to 110 ℃ for more than 10 hours, characterized in that: adding a fertilizer in the presence of an ammonium salt acting as glucosamine precursor during the cultivation of a plant selected from the group of plant species comprising sucrose, fructose or inulin prior to harvesting.

2. The method of claim 1, wherein the fertilizer is a compound capable of forming a sugar-nitrogen compound condensation reaction necessary for glucosamine production.

3. The process according to claim 1 or 2, wherein the plant species belongs to the genus Cichorium, Daucus, Helianthus and/or beta.

4. A process according to claim 1 or 2, wherein the plant is chicory, carrot, Jerusalem artichoke and/or beet.

5. Process according to claim 4 wherein chicory is produced under hydroponic conditions.

6. Process according to claim 5, wherein a fertilizer is added to the nutrient solution of the hydroponic chicory root before or after the production of endives.

7. Process according to claim 6, wherein the roots obtained from the production of endives are used as source of glucosamine.

8. Plant material or plant extract obtained from the method of any one of claims 1 to 7, containing at least 5g glucosamine/kg dry weight of plant material.

9. Plant material or plant extract according to claim 8, containing at least 7.5g glucosamine/kg dry weight of plant material.

10. Plant material or plant extract according to claim 8, containing more than 10g glucosamine/kg dry weight of plant material.

11. Plant material or plant extract according to claim 8, wherein at least part of the glucosamine is in free form.

12. Plant material or plant extract according to claim 8, wherein at least half of the glucosamine is in free form.

13. Plant material or plant extract according to any one of claims 8 to 12, wherein the plant belongs to the genus cichorium, daucus, sunflower or beta.